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Discovery of a mRNA mitochondrial localization element in Saccharomyces cerevisiae by nonhomologous random recombination and in vivo selection.

Liu JM, Liu DR - Nucleic Acids Res. (2007)

Bottom Line: To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3' UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization.Site-directed mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity.In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 01238, USA.

ABSTRACT
In budding yeast, over 100 nuclear-encoded mRNAs are localized to the mitochondria. The determinants of mRNA localization to the mitochondria are not well understood, and protein factors involved in this process have not yet been identified. To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3' UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization. When applied to the yeast ATP2 mRNA, this approach rapidly identified a 50-nt consensus motif, designated Min2, as well as two Min2-homologous regions naturally present downstream of the ATP2 stop codon, which are sufficient when appended to the 3' end of various reporter mRNAs to induce mitochondrial localization. Site-directed mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity. In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.

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Mutagenesis analysis of Min2. The sequence and predicted secondary structure (31,32) of Min2 are shown above. Active (green) and significantly impaired (red) mutants are shown. Activity values shown reflect the mean fraction of colonies surviving on glycerol medium relative to wild-type (pATP2 carrying the native 636 nt ATP2 3′ UTR); control is a 500 nt random sequence. Nucleotides 11, 12 and 27–32 (highlighted in orange) were deleted in mutant M5. Error values reflect the SD of three or more independent trials. Bold nucleotides are highly conserved among the 13 sequences subjected to MEME analysis. Italicized nucleotides are non-conserved.
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Figure 5: Mutagenesis analysis of Min2. The sequence and predicted secondary structure (31,32) of Min2 are shown above. Active (green) and significantly impaired (red) mutants are shown. Activity values shown reflect the mean fraction of colonies surviving on glycerol medium relative to wild-type (pATP2 carrying the native 636 nt ATP2 3′ UTR); control is a 500 nt random sequence. Nucleotides 11, 12 and 27–32 (highlighted in orange) were deleted in mutant M5. Error values reflect the SD of three or more independent trials. Bold nucleotides are highly conserved among the 13 sequences subjected to MEME analysis. Italicized nucleotides are non-conserved.

Mentions: These clones were chosen for the following reasons: first, they provided a range in sequence length, ranging from 60 to 238 nt; second, they collectively represented random-derived, sense-derived and antisense-derived active sequences; lastly, these sequences exhibited positive phenotypes in both the glycerol and gRNA screens. The full-length sequences of all 13 samples are listed in Table 2. The MEME analysis identified motifs commonly found in active clones. One conserved motif, a 50-nt sequence designated Min2 (Figure 5), emerged as having by far the greatest statistical significance (lowest probability of arising randomly). The sequence logo of Min2, depicting the relative contribution of each of the original 13 sequences towards generating the minimal motif, is depicted in Figure 6 (29,30).Figure 5.


Discovery of a mRNA mitochondrial localization element in Saccharomyces cerevisiae by nonhomologous random recombination and in vivo selection.

Liu JM, Liu DR - Nucleic Acids Res. (2007)

Mutagenesis analysis of Min2. The sequence and predicted secondary structure (31,32) of Min2 are shown above. Active (green) and significantly impaired (red) mutants are shown. Activity values shown reflect the mean fraction of colonies surviving on glycerol medium relative to wild-type (pATP2 carrying the native 636 nt ATP2 3′ UTR); control is a 500 nt random sequence. Nucleotides 11, 12 and 27–32 (highlighted in orange) were deleted in mutant M5. Error values reflect the SD of three or more independent trials. Bold nucleotides are highly conserved among the 13 sequences subjected to MEME analysis. Italicized nucleotides are non-conserved.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175308&req=5

Figure 5: Mutagenesis analysis of Min2. The sequence and predicted secondary structure (31,32) of Min2 are shown above. Active (green) and significantly impaired (red) mutants are shown. Activity values shown reflect the mean fraction of colonies surviving on glycerol medium relative to wild-type (pATP2 carrying the native 636 nt ATP2 3′ UTR); control is a 500 nt random sequence. Nucleotides 11, 12 and 27–32 (highlighted in orange) were deleted in mutant M5. Error values reflect the SD of three or more independent trials. Bold nucleotides are highly conserved among the 13 sequences subjected to MEME analysis. Italicized nucleotides are non-conserved.
Mentions: These clones were chosen for the following reasons: first, they provided a range in sequence length, ranging from 60 to 238 nt; second, they collectively represented random-derived, sense-derived and antisense-derived active sequences; lastly, these sequences exhibited positive phenotypes in both the glycerol and gRNA screens. The full-length sequences of all 13 samples are listed in Table 2. The MEME analysis identified motifs commonly found in active clones. One conserved motif, a 50-nt sequence designated Min2 (Figure 5), emerged as having by far the greatest statistical significance (lowest probability of arising randomly). The sequence logo of Min2, depicting the relative contribution of each of the original 13 sequences towards generating the minimal motif, is depicted in Figure 6 (29,30).Figure 5.

Bottom Line: To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3' UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization.Site-directed mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity.In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 01238, USA.

ABSTRACT
In budding yeast, over 100 nuclear-encoded mRNAs are localized to the mitochondria. The determinants of mRNA localization to the mitochondria are not well understood, and protein factors involved in this process have not yet been identified. To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3' UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization. When applied to the yeast ATP2 mRNA, this approach rapidly identified a 50-nt consensus motif, designated Min2, as well as two Min2-homologous regions naturally present downstream of the ATP2 stop codon, which are sufficient when appended to the 3' end of various reporter mRNAs to induce mitochondrial localization. Site-directed mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity. In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.

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